Taqman snp genotyping

Manufactured by Thermo Fisher Scientific

Sourced in United States, Singapore

TaqMan SNP genotyping is a real-time PCR-based assay that enables the detection and identification of specific single nucleotide polymorphisms (SNPs) in a DNA sample. The TaqMan assay utilizes fluorescent probe technology to accurately and efficiently determine the genotype of a target SNP.

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Lab products found in correlation

Dna blood midi kit, by Qiagen (2 mentions) Lightsnip, by TIB Molbiol (1 mentions) Dna isolation kit, by Qiagen (1 mentions) Lightcycler 480 2, by Roche (1 mentions) Ds 11, by DeNovix (1 mentions) Abi prism 3130xl sequencer, by Thermo Fisher Scientific (1 mentions) Taqman pre designed snp genotyping assay, by Thermo Fisher Scientific (1 mentions) Bigdye terminator cycle sequencing kit version 1, by Thermo Fisher Scientific (1 mentions) Maldi tof, by Labcorp (1 mentions) Quant it picogreen assay, by Thermo Fisher Scientific (1 mentions) Type it fast snp probe pcr kit, by Qiagen (1 mentions) Qiaamp dna blood kit, by Qiagen (1 mentions) Dneasy blood and tissue kit, by Qiagen (1 mentions) Qiaquick pcr purification kit, by Qiagen (1 mentions) Abi prism 7000 system, by Thermo Fisher Scientific (1 mentions) Steponeplus, by Thermo Fisher Scientific (1 mentions) Restriction enzyme digestion, by New England Biolabs (1 mentions) Sequence detection system 2, by Thermo Fisher Scientific (1 mentions)

24 protocols using taqman snp genotyping

Genetic Profiling of Essential Tremor

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Consecutive patients with ET who presented to tertiary referral centres and examined by movement disorders neurologists were included. Patients satisfied the diagnostic criteria of classic ET based on the recommendations of the Consensus Statement of the Movement Disorders Society4 (link). Healthy individuals of similar gender, race and age were included as controls. Subjects with evidence of other neurodegenerative diseases were excluded. ET cases did not have evidence of thyroid dysfunction. Written informed consent from all the study subjects was obtained.
Genomic DNA was extracted from blood samples and LRRK2 c.4883G>C (R1628P; rs33949390) was genotyped by Taqman SNP genotyping (Life Technologies, Singapore). All positives and selected negatives were further confirmed by capillary/Sanger sequencing analysis with standard protocols. Our study was approved by the SingHealth Centralised Institutional Review Board (CIRB) and all the methods were carried out in accordance with the approved guidelines. Fisher's Exact Test for Count Data was used to compare the categorical and numerical variables. Statistical significance was defined at p < 0.05.

Chao Y.X., Ng E.Y., Tan L., Prakash K.M., Au W.L., Zhao Y, & Tan E.K. (2015). Lrrk2 R1628P variant is a risk factor for essential tremor. Scientific Reports, 5, 9029.

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LRRK2 R1628P Genotyping Protocol

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Blood samples were collected from all participants for genotyping. DNA was extracted from the blood samples collected using Blood DNA Midi Kit (Qiagen, USA). LRRK2 c.4883aG > C (R1628P; rs33949390) was genotyped by Taqman SNP genotyping (Life Technologies, Singapore). All positives and selected negatives were confirmed by capillary/Sanger sequencing analysis.

Kumar P.M., Paing S.S., Li H., Pavanni R., Yuen Y., Zhao Y, & Tan E.K. (2015). Differential effect of caffeine intake in subjects with genetic susceptibility to Parkinson’s Disease. Scientific Reports, 5, 15492.

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Genetic Profiling from Blood Samples

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Blood samples were collected from subjects and genotyped. DNA extraction was carried out using Blood DNA Midi Kit (Qiagen, USA) and genotyped by Taqman SNP genotyping (Life Technologies, Singapore). All positives and selected negatives were confirmed by capillary/Sanger sequencing analysis with standard protocol.

Ong Y.L., Deng X., Li H.H., Narasimhalu K., Chan L.L., Prakash K.M., Au W.L., Ratnagopal P., Tan L.C, & Tan E.K. (2023). Caffeine intake interacts with Asian gene variants in Parkinson's disease: a study in 4488 subjects. The Lancet Regional Health: Western Pacific, 40, 100877.

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Genetic Profiling of AML Patients

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Genomic DNA was extracted from peripheral blood taken on EDTA from 92 AML patients and 135 healthy individuals using a Qiagen DNA Isolation Kit (QIAGEN, Hilden, Germany). DNA was also isolated from AML cell lines. DNA purity and concentration were verified on the DeNovix DS-11 spectrophotometer (DeNovix Inc., Wilmington, DE, USA). Isolated DNA was subsequently stored at −20 °C until further use.
BSG and MCT1 SNPs were selected based on three criteria: (1) minor allele frequency (MAF) in European populations higher than 0.15, (2) a functional effect on expression/protein structure predicted by the National Institute of Environmental Health Sciences SNP Function Prediction tool [36 (link)], and (3) lack of high linkage disequilibrium between the SNPs. Based on these criteria, six SNPs (four in the gene coding for BSG and two in the gene coding for MCT1) were chosen. BSG and MCT1 SNP were determined using TaqMan SNP Genotyping (Applied Biosystems, Waltham, MA, USA) and LightSNiP (TIB MOLBIOL, Berlin, Germany) assays. PCR was performed on a LightCycler 480 II (Roche Diagnostics, Rotkreuz, Switzerland) according to the manufacturers’ protocols.

Łacina P., Butrym A., Turlej E., Stachowicz-Suhs M., Wietrzyk J., Mazur G, & Bogunia-Kubik K. (2022). BSG (CD147) Serum Level and Genetic Variants Are Associated with Overall Survival in Acute Myeloid Leukaemia. Journal of Clinical Medicine, 11(2), 332.

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SNP Genotyping from Peripheral Blood

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Genomic DNA was extracted from peripheral blood leukocytes according to established protocols. Genotyping of SNPs was carried out by direct sequencing, TaqMan SNP genotyping (Applied Biosystems, Foster City, USA) or by primer extension of multiplex PCR products and subsequent allele detection by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF; Sequenom, San Diego, USA). Direct sequencing was performed with the Big Dye Terminator Cycle sequencing kit version 1.1 (Applied Biosystems, Foster City, U.S.A.) according to the manufacturer’s instructions. Reactions were analyzed with an ABI Prism 3130xl sequencer (Applied Biosystems). TaqMan pre-designed SNP genotyping assays (Applied Biosystems) were used according to the manufacturer’s instructions. The rs144087548 variant was genotyped by polymerase chain reaction (forward primer: 5′-CGC AGA CAT GAT GCT GGG GGT-3′; reverse primer: 5′-ACA TGC AAG ACG GGG AAT TGA-3′) followed by HpyCH4III digestion (New England Biolabs, Ipswich, USA) and restriction fragment length analysis. All SNPs showed high genotyping quality with an average call rate >98 % in each of the five case–control samples.

Grassmann F., Friedrich U., Fauser S., Schick T., Milenkovic A., Schulz H.L., von Strachwitz C.N., Bettecken T., Lichtner P., Meitinger T., Arend N., Wolf A., Haritoglou C., Rudolph G., Chakravarthy U., Silvestri G., McKay G.J., Freitag-Wolf S., Krawczak M., Smith R.T., Merriam J.C., Merriam J.E., Allikmets R., Heid I.M, & Weber B.H. (2015). A Candidate Gene Association Study Identifies DAPL1 as a Female-Specific Susceptibility Locus for Age-Related Macular Degeneration (AMD). Neuromolecular Medicine, 17(2), 111-120.

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NOD2 Genotyping in Healthy Individuals

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Informed consent was obtained per protocol approved by Yale University IRB. We performed genotyping by TaqMan SNP genotyping (Applied Biosystems, Foster City, CA) or Sequenom platform (Sequenom Inc., San Diego, CA). We utilized cells from healthy individuals not homozygous for R702W, G908R or Leu1007insC NOD2 mutations.

Lahiri A, & Abraham C. (2014). Activation of Pattern Recognition Receptors Upregulates Metallothioneins, Thereby Increasing Intracellular Accumulation of Zinc, Autophagy, and Bacterial Clearance by Macrophages. Gastroenterology, 147(4), 835-846.

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SNP Genotyping Using TaqMan Assays

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De novo genotyping of the SNP rs7806429 was done using the TaqMan SNP Genotyping assays according to the manufacturer's protocol (Applied Biosystems, Inc., Foster City, CA). To validate the reproducibility of genotyping, a random subset (about 5%) of the sample was re-genotyped; all genotypes matched initial designated genotypes. No deviations from HWE were observed (p>0.1).

Tönjes A., Scholz M., Breitfeld J., Marzi C., Grallert H., Gross A., Ladenvall C., Schleinitz D., Krause K., Kirsten H., Laurila E., Kriebel J., Thorand B., Rathmann W., Groop L., Prokopenko I., Isomaa B., Beutner F., Kratzsch J., Thiery J., Fasshauer M., Klöting N., Gieger C., Blüher M., Stumvoll M, & Kovacs P. (2014). Genome Wide Meta-analysis Highlights the Role of Genetic Variation in RARRES2 in the Regulation of Circulating Serum Chemerin. PLoS Genetics, 10(12), e1004854.

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Genotyping of Doxorubicin-Associated Variants

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Genomic DNA was isolated from EDTA blood samples with the QIAamp DNA Blood Kit (Qiagen). Saliva was collected with the Oragene DNA collection kit (DNA Genotec), and genomic DNA was extracted from saliva samples using the prepIT L2P reagent (DNA Genotec). DNA was quantified by Quant-iT PicoGreen assay (Invitrogen), according to the manufacturer’s protocols. Genomic DNA samples were genotyped for RARG rs2229774, SLC28A3 rs7853758, and UGT1A6 rs17863783 (Table 2) by TaqManSNP genotyping (Applied Biosystems), using the Type-it Fast SNP Probe PCR Kit (Qiagen) with predesigned primers and probes (Applied Biosystems).
Laboratory assistants were blinded to the case-control status of the patients genotyped in the study. To ensure the accuracy of all genotyping results, multiple positive and negative controls and replicate samples were included in all genotyping assays and plates. The concordance of genotype calls between replicate genotyped samples was 100%.
Genomic DNA will also be used to replicate the association of doxorubicin-related cardiovascular toxicity with other candidate genes that have been described [22 (link)] or that will be discovered in the future, provided our sample size is sufficient.

Zolk O., von dem Knesebeck A., Graf N., Simon T., Hero B., Abdul-Khaliq H., Abd El Rahman M., Spix C., Mayer B., Elsner S., Gebauer J, & Langer T. (2022). Cardiovascular Health Status And Genetic Risk In Survivors of Childhood Neuroblastoma and Nephroblastoma Treated With Doxorubicin: Protocol of the Pharmacogenetic Part of the LESS-Anthra Cross-Sectional Cohort Study. JMIR Research Protocols, 11(2), e27898.

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Genotyping PTPN22 and HLA-A from Blood

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DNA extraction from whole blood was performed using the DNeasy Blood and Tissue kit (Qiagen). PTPN22 C1858T genotype on all subjects was determined by TaqMan SNP Genotyping (Applied Biosystems). HLA-A genotype was determined through PCR amplification [32 (link)], purification of PCR product using QIAquick PCR Purification Kit (Qiagen), and Sanger sequencing. HLA-A type was determined by BLAST of PCR sequence against the IMGT/HLA database.

Tien S.H., Crabtree J.N., Gray H.L, & Peterson E.J. (2017). Immunologic response to vaccine challenge in pregnant PTPN22 R620W carriers and non-carriers. PLoS ONE, 12(7), e0181338.

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Inflammatory Bowel Disease Genetics

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Informed consent was obtained per protocol approved by the institutional review board at Yale University. For cell studies in healthy controls, we recruited participants with no personal or family history of autoimmune/inflammatory disease, including psoriasis, SLE, rheumatoid arthritis, multiple sclerosis, type I diabetes mellitus, CD, and ulcerative colitis, or a history of HIV. Due to the limitation in primary human cells and the range of stimulation conditions we wished to interrogate, two separate cohorts of 100 and 98 individuals were recruited for NOD2 and TLR2 dose-response studies, and NOD2 and TLR synergy studies in MDDC, respectively. Genotyping for polymorphisms was performed by TaqMan SNP genotyping (Applied Biosystems, Foster City, CA) or Sequenom platform (Sequenom Inc., San Diego, CA). For association studies, a total of 2724 subjects were enrolled, including 1512 CD cases (542 ileal, 705 ileocolonic and 265 colonic cases) and 1212 healthy controls. CD patients were diagnosed by clinical, endoscopic, radiologic and histologic criteria. Medical histories were collected with the validated NIDDK IBD Genetics Consortium phenotype forms (Nguyen et al., 2006 (link)).

Hedl M., Lahiri A., Ning K., Cho J.H, & Abraham C. (2014). Pattern Recognition Receptor Signaling in Human Dendritic Cells is Enhanced by ICOS Ligand and Modulated by the Crohn’s Disease ICOSLG Risk Allele. Immunity, 40(5), 734-746.

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